DOI: 10.1002/cbic.200500041 Nanostructured Ordering of Fluorescent Markers and Single Proteins on Substrates Juergen Groll,* [a] Krystyna Albrecht, [a] PeterGasteier, [a] SilkeRiethmueller, [b] UlrichZiener, [b] and Martin Moeller* [a] Introduction Immobilization of proteins on surfaces in ordered patterns is a very active field of research. [1–3] In contrast to DNA chips, where the general shape, charge, immobilization chemistry, and detection method are essentially the same for the various oligonucleotides, proteins differ considerably in all these prop- erties. Therefore, the prevention of unspecific interactions and the specific immobilization of different proteins on the molec- ular level in their native and functional conformations are chal- lenging tasks. [4–6] Technically, despite possibilities like ink-jet technology, [7] photolithography, [8] selective self-assembly, [9] or microcontact printing, [10–12] the production of microarrays is mainly achieved by spotting techniques. [13,14] Attempts to pro- duce patterns with resolutions in the submicron range are harder to realize. Microcontact printing has been used to gen- erate protein patterns with single-protein resolution, [15] but the regularity of the pattern is not perfect. Dip-pen nanolithogra- phy [16–18] isapromisingmethodbutitislimitedtothepattern- ingofverysmallsampleareas.Thegenerationofregularnano- patterns over large surface areas can be accomplished by self- assembly, for example, of block-copolymer micelles on sub- strates. [19,20] This method enables the generation of gold nano- clusters on substrates in regular hexagonal patterns. [21–23] The applicability of such patterns for biological studies has recently been demonstrated by Spatz and co-workers, who used the gold dots as anchoring points for the specific attachment of cells. [24] Fluorescent molecules adsorbed on metallic surfaces exhibit strong quenching of their fluorescence due to electromagnetic coupling between the metal and the dye molecules. [25,26] Inad- dition, fluorescence dyes at metal surfaces change their fluo- rescence rate, their fluorescence lifetime, and their fluores- cence yield. [27–29] This process has been studied extensively for metal films, but the situation differs for nanoparticles. Two de- cisive parameters are the size and geometry of the particles and the distance between the dye molecule and the particle. Whereas a lissamine dye coupled to a gold nanoparticle by a thioether group at a distance of 1nm exhibits pronounced fluorescence quenching, [30] the fluorescence of the same dye with a longer thioether spacer, which keeps the dye–nanopar- ticle distance at 2nm when attached to gold nanoparticles on a solid substrate, resulted in an easily detectable fluorescence intensity. [31] In other work, an unsymmetrical bis(perylene bis- imide) dye bearing a disulfide functional group undergoes self- assembly to form aggregated structures on gold surfaces. In- tense fluorescence from these dye aggregates is observed on surfaces decorated with hexagonal gold patterns, whereas the fluorescenceisonlyweakonplaingoldsubstrates. [32] The present study shows the binding of fluorescence dyes and proteins to highly ordered gold-nanodot arrays on sub- strate surfaces. Since the arrays are prepared by dip-coating into micellar block-copolymer solutions, large sample areas can be patterned easily. The selective immobilization of nitrilotri- acetic acid (NTA) groups to the gold dots was visualized by complex formation with nickel(ii )chlorideandthefluorescence dye Newport Green. To prevent unspecific dye adsorption in this case, the substrate between the gold dots was modified by a hydrophobic octadecyltrichlorosilane (OTS) self-assembled Highly ordered hexagonal nanopatterns of gold clusters on glass substrates were used as anchoring points for the specifc attach- ment of fluorescence dyes and proteins labeled with fluorescence dyes. Thiol- or disulfide-containing linker molecules were used for the binding to the gold dots. In order to ensure specific binding on the gold dots only, the surface area in between the dots was protected against unspecific adsorption. For the attachment of polar low-molecular-weight fluorescence dyes, an octadecyltri- chlorosilane self-assembled monolayer was prepared on the sur- face in between the gold dots, whereas a layer prepared from star-shaped poly(ethylene oxide-stat-propylene oxide) prepoly- mers was used to prevent unspecific adsorption of proteins be- tween the gold dots. Fluorescence microscopy proved the specific binding of the dyes as well as of the proteins. Scanning force mi- croscopy studies show that each gold dot is only capable of bind- ing one protein at a time. [a] Dr. J. Groll, K. Albrecht, P. Gasteier, Prof. Dr. M. Moeller Deutsches Wollforschungsinstitut an der RWTH Aachen e.V. Pauwelsstrasse 8, 52074 Aachen (Germany) Fax: (+ 49)241-802-3301 E-mail: groll@dwi.rwth-aachen.de moeller@dwi.rwth-aachen.de [b] Dr. S. Riethmueller, Dr. U. Ziener Department of Organic Chemistry III–Macromolecular Chemistry University of Ulm Albert Einstein Allee 11, 89081 Ulm (Germany) Supporting information for this article is available on the WWW under http://www.chembiochem.org or from the author. ChemBioChem 2005,6,1–7 #2005Wiley-VCHVerlagGmbH&Co.KGaA,Weinheim 1